Method of treating neuronal injury by administering magnesium chloride and PEG

ABSTRACT

The invention provides methods and kits for treatment of pain or inflammation. In one embodiment, the kit comprises a biomembrane sealing agent, such as PEG, and a bioactive agent, such as a magnesium compound. The biomembrane sealing agent and/or the bioactive agent an intravenous administration, an intramuscular administration, an intrathecal administration, a subcutaneous administration, an epidural administration, a parenteral administration, an intra-articular administration, a direct application onto or adjacent to a site of the pathological condition, and any combinations thereof. Alternatively, the biomembrane sealing agent and/or the bioactive agent may be delivered from a pump or an implant.

FIELD OF THE INVENTION

This invention relates to methods and composition of treating conditionsassociated with pain or inflammation.

BACKGROUND

Clinical indications affecting the central nervous system such astraumatic brain injury (TBI), spinal cord injury (SCI) and stroke areleading causes of mortality and morbidity in the industrialized world.For example, about 1 million of Americans per year are treated for braininjury in emergency rooms. Approximately 5% of the TBI patients die and30% of the survivors are generally left with moderate to severedisabilities that may impair their ability to return to work or liveindependently. Following neuronal injury, a significant proportion ofpatients will also develop chronic painful conditions.

Pain in general is associated with a myriad of medical conditions andaffects millions of Americans. As reported by the American PainFoundation, over 50 million Americans suffer from chronic pain including20% of individuals aged 60 and over who are affected by joint disorderssuch as arthritis. Furthermore, nearly 25 millions Americans experienceacute pain due to injuries or surgical procedures each year. In additionto its economical burden, pain has a tremendous effect on the quality oflife of affected individuals and is one of the most common causes ofdisability.

Accordingly, novel improved methods and compositions of treating pain orinflammation are desired to alleviate these debilitating conditions.

SUMMARY OF INVENTION

The instant invention fulfills this and the other foregoing needs byproviding novel kits and methods for treatment of conditions associatedwith pain or inflammation. In one aspect, the present invention providesa kit for treating a pathological condition associated with pain orinflammation comprising at least one biomembrane sealing agent, at leastone bioactive agent and a set of instructions comprising information onmaking an injectable composition, comprising more than about 10% of theat least one biomembrane sealing agent. In one embodiment of theinvention, the composition is incapable of forming a gel.

In different embodiments of the invention, the at least one biomembranesealing agent is selected from the group consisting of polyoxyethylenes,polyalkylene glycol, polyethylene glycol or PEG, polyvinyl alcohol,pluronics, poloxamers, methyl cellulose, sodium carboxylmethylcellulose, hydroxyethyl starch, polyvinyl pyrrolidine, dextrans,poloxamer P-188, and any combinations thereof.

The at least one bioactive agent is selected from the group consistingof at least one magnesium compound, antioxidants, neurotransmitter andreceptor modulators, anti-inflammatory agents, anti-apoptotic agents;nootropic and growth agents; modulators of lipid formation andtransport; blood flow modulators; electrical stimulation; and anycombinations thereof.

In other embodiments, the at least one bioactive compound comprises atleast one inosine, dexanabinol, electrical or magnetic stimulation, CP101,606, RPR117824, CD11b/CD18 antibody, CD95 Blocker, ATL-146e, CM101,Riluzole, Topiramate, Amantadine, Gacyclidine, BAY-38-7271, S-1749,YM872, IL-1, IL-8 and TNF-alpha blockers, IL-10, DFU, NXY-059,Edaravone, N-tert-butyl-alpha-phenylnitrone, glutathione and derivates,Rho kinase inhibitors, erythropoietin, steroids, statins IGF-1, GDNF,choline or CDP-choline, creatine, AIT-082, Cyclosporine A, FK-506,Minocycline, Triamcinolone, Methylprednisolone or any combinationthereof.

In different embodiments, the at least one magnesium compound comprisesmagnesium sulfate, magnesium chloride, magnesium gluconate, magnesiumATP, or any combination thereof.

In yet another aspect, the invention provides a method of treating apathological condition associated with pain or inflammation, the methodcomprising delivering to a subject in need thereof a therapeuticallyeffective amount of at least one biomembrane sealing agent and atherapeutically effective amount of at least one bioactive agent,wherein the at least one biomembrane sealing agent is delivered in aninjectable composition, wherein the at least one biomembrane sealingagent comprises more than about 10% of the injectable composition. Inone embodiment of the invention, the composition is incapable of forminga gel. Further, in different embodiments, the at least one biomembranesealing agent and the at least one bioactive agent may be delivered by amethod selected from the group consisting of an intravenousadministration, an intramuscular administration, an intrathecaladministration, a subcutaneous administration, an intra-articularadministration, an epidural administration, a parenteral administration,a direct application onto a site of the pathological condition, animplanted depot, and any combinations thereof.

DETAILED DESCRIPTION

The instant invention provides novel kits and methods for treatment ofconditions associated with pain or inflammation. The discovery of asynergistic effect between PEG, a biomembrane sealing agent, andmagnesium is highly significant as it can lead to the development oftherapeutic formulations with improved efficacy for the treatment ofinflammation and painful conditions.

Definitions

To aid in the understanding of the invention, the following non-limitingdefinitions are provided:

The term “treating” or “treatment” of a disease refers to executing aprotocol, which may include administering one or more drugs to a patient(human or otherwise), in an effort to alleviate signs or symptoms of thedisease. Alleviation can occur prior to signs or symptoms of the diseaseappearing, as well as after their appearance. Thus, “treating” or“treatment” includes “preventing” or “prevention” of disease. Inaddition, “treating” or “treatment” does not require completealleviation of signs or symptoms, does not require a cure, andspecifically includes protocols which have only a marginal effect on thepatient.

The term “subject” includes a living or cultured system upon which themethods and/or kits of the current invention is used. The term includes,without limitation, humans.

The term “practitioner” means a person who practices methods, kits, andcompositions of the instant invention on the subject. The term includes,without limitations, doctors, other medical personnel, and researchers.

The terms “neuropathic pain” and “neural origin pain” refer to paininitiated or caused by a pathological condition of the nervous system,including, without limitation, pathology following chronic or acuteinsults.

The hallmarks of neuropathic pain are chronic allodynia andhyperalgesia. Accordingly, the term “allodynia” refers to pain resultingfrom a stimulus that ordinarily does not elicit a painful response.

The term “hyperalgesia” refers to an increased sensitivity to a normallypainful stimulus. Primary hyperalgesia affects the immediate area of theinjury.

The term “secondary hyperalgesia” or “referred pain” is normallyutilized in cases when sensitization has extended to a broader areasurrounding the injury.

The term “neuronal injury” refers to an insult to an element of thecentral or peripheral nervous systems. Neuronal injuries can be derivedfrom a physical (including mechanical, electrical or thermal), ischemic,hemorrhagic, chemical, biological or biochemical insult. Examples ofneuronal injuries include, without limitations, ischemic and hemorrhagicstroke, spinal cord, brain, cranial nerve and peripheral nerve injuries.

The term “bioactive agent” refers to molecules and physical stimuli.

All references to chemical compounds, including without limitation,bioactive agents, biomembrane sealing agents, and markers, include allforms of these chemical compounds (i.e., salts, esters, hydrates,ethanolates, etc.), wherein said forms possess at least partialactivities of the respective chemical compounds.

There are two basic forms of physical pain: acute and chronic. Acutepain, for the most part, results from disease, inflammation, or injuryto tissues. It is mediated by activation of sensory fibers also known asnociceptive neurons. Nociceptive pain normally disappears after healing,for example in cases of post-traumatic or post-operative pain.Unfortunately, in some individuals, pathological changes occur thatincrease the sensitivity of the sensory neurons. In those cases,symptomatic pain can become chronic and persists for months or evenyears after the initial insult.

Neuronal injuries are complex clinical conditions aggravated by avariety of precipitating causes that influence the severity of injuryand ultimately influence the course and extent of recovery. A primaryinsult to a component of the central and/or peripheral nervous systemcould be of mechanical, chemical, biological or electrical nature.Following the primary insult, a cascade of biochemical and physiologicalevents takes place that often leads to pathobiological changes that areconsidered largely responsible for the development of irreversibledamages. This autodestructive cascade is known as secondary injury andbecause it develops over time after the traumatic event it opens awindow of opportunity for pharmacological interventions. Various chronicconditions linked to persistent on-going tissue damage due, for example,to inflammatory reactions or autoimmune diseases, may also lead tosecondary injury of neuronal components and symptomatic pain.

There are at least three major classes of events that are determinant inthe secondary phase of the traumatic brain injury (TBI) pathology andother neuronal injuries. One of these is membrane damages to cells thatmanaged to survive the first impact. Small changes in membrane integrityand/or cytoskeletal architecture can impair membrane potential,intracellular transport and ATP production leading to cytoskeletalcollapse, mitochondrial dysfunction, energetic failure and free radicalproduction resulting in cell death by either apoptotic or necroticmechanisms. In some instances, the dying cells can release free radicalsand catabolic enzymes (proteinase, peptidases, caspases) that may causedamages to the surrounding cells and increase the number of injuredcells. Unfortunately, neuronal cells are particularly vulnerable tomembrane damages due to their high energetic demand and their uniqueanatomical structure which increase by many folds the challenge ofmaintaining efficient membrane integrity and intracellular (axonal)transport.

The other class of events that plays a major role in the secondary phaseof neuronal injuries is the storm of neurotransmitter release which bymechanisms globally referred as “excitotoxicity” can increase thevulnerability of neurons to any additional insults in an area that, insize, extend far beyond the area directly affected by the first insult.For example, a marked increase in extracellular glutamate levels isoften associated with neuronal insults. Since glutamate is the mostprominent excitatory neurotransmitter of the central nervous system,almost all neurons have glutamate receptors and will be affected by thetoxic events triggered by excessive amounts of extracellular glutamate.Excitotoxicity insults are thought to be largely triggered by excessiveinflow of Ca²⁺ through a specific subtype of glutamate receptor, theN-methyl-D-aspartate receptors (NMDAR). High concentrations ofintracellular Ca²⁺ can activate catabolic enzymes and production of freeradicals that may interfere with the repair mechanisms of the cell orits ability to cope with additional challenges or even precipitate celldeath.

The third class of detrimental events is linked to vascular damages andbreakdown of the blood-brain barrier. In animal models of TBI and SCI,the blood-brain barrier remains disrupted for many days after injury(Schnell et al., 1999) allowing for extravasation of plasma proteins andinvasion of the central nervous system (CNS) by blood and immune cells.

Although the role of inflammation in brain injury and other forms ofneuronal injuries remains controversial, proper distribution ofnutrients to the nervous tissue cannot be accomplished before theblood-brain barrier and the cerebral vessels are repaired.

Inflammation is the body's normal protective response to conditions thatinclude a tissue necrosis component. Tissue necrosis can be derived froma physical (including mechanical, electrical or thermal), chemical,biological or biochemical insult. Clinical conditions with aninflammatory component include traumatic tissue injury, surgery,degenerative diseases such as arthritis and other joint diseases as wellas irritation, hypersensitivity, and auto-immune reactions.

During this natural “defense” process, local increases in blood flow andcapillary permeability lead to accumulation of fluid, proteins andimmune cells in the inflamed area. Some of these cells can releasechemical mediators of inflammation including histamine, cytokines,bradykinin and prostaglandins that can attract more immune cells at thesite of inflammation and/or increase the sensitivity of pain fiberswithin the affected area. As the body mounts this protective response,the symptoms of inflammation develop. These symptoms include, withoutlimitation, pain, swelling and increased warmth and redness of the skin.The inflammatory response has to be tightly regulated otherwise it maylead to tissue necrosis and development of chronic pain.

Biomembrane Sealing Agents

For more than 40 years, biomembrane sealing agents of various molecularweights have been utilized as adjuncts to culture media for theirability to protect cells against fluid-mechanical injuries. These agentsinclude hydrophilic polymers such as polyoxyethylenes, polyalkyleneglycol, polyethylene glycols (PEG), polyvinyl alcohol, amphipaticpolymers such as pluronics or poloxamers, including poloxamer P-188(also known as CRL-5861, available from CytRx Corp., Los Angeles,Calif.) (Michaels and Papoutsakis, 1991) as well as methyl cellulose(Kuchler et al., 1960), sodium carboxylmethyl cellulose, hydroxyethylstarch, polyvinyl pyrrolidine and dextrans (Mizrahi and Moore, 1970;Mizrahi, 1975; Mizrahi, 1983).

Some biomembrane sealing agents including hydroxyethyl starch (Badet etal., 2005) and PEG (Faure et al, 2002; Hauet et al., 2001) have showneffective cryopreservative abilities in organ transplantation studies.Poloxamer P-188 was shown to protect articular cells from secondaryinjury following mechanical trauma to knee joint which could lead toacute pain and inflammation and potentially develop into a more chroniccondition known as osteoarthritis (Phillips and Haut, 2004). PoloxamerP-188 and a neutral dextran protected muscle cells againstelectroporation or thermally driven cell membrane permeabilization (Leeet al., 1992). Direct application of PEG was shown to anatomically andfunctionally reconnect transected or crushed axon (Bittner et al.,1986), peripheral nerve (Donaldson et al., 2002) and spinal cordpreparations in vitro (Lore et al., 1999; Shi et al., 1999; Shi andBorgens, 1999; Shi and Borgens, 2000; Luo et al., 2002) or in vivo(Borgens et al., 2002). Intravenous or subcutaneous administration ofPEG or Poloxamer P-188 improved the cutaneous trunchi muscle reflexresponse after experimental spinal cord contusion in guinea pigs(Borgens and Bohnert, 2001; Borgens et al., 2004) and improvedfunctional recovery in a naturally occurring spinal cord injury model indogs (Laverty et al., 2004). PEGs of various molecular weights from1,400-20000 Da, having a linear or multiple arms structure were shown toimprove recovery following tissue injury (Hauet et al., 2001; Detloff etal., 2005; Shi et al., 1999).

Biomembrane sealing agents can be effective following different modes ofdelivery including local and prolonged cellular exposure, direct andshort-term tissue or organ exposure or systemic administration.Effective concentrations of biomembrane fusion agents may vary dependingon the purpose and/or mode of delivery. For example, about 0.05%concentration is effective in tissue culture applications (Michaels andPapoutsakis, 1991) and about 30% to about 50% concentration is effectivefor organ preservation and upon in vivo administration in animals (Hauetet al., 2001; Shi et al., 1999; Borgens and Bohnert, 2001; Borgens etal., 2004).

Bioactive Agents

As discussed above, inventors have found that a combination of abiomembrane sealing agent and a magnesium compound is useful for thetreatment of neuronal trauma and painful conditions. Accordingly, in oneembodiment of the invention, the at least one bioactive agent comprisesa magnesium compound. Magnesium plays an important role in a largediversity of cellular functions. For example, magnesium is required forglycolysis and oxidative phosphorylation which support energy-producingand energy-consuming reactions in cells. Protein synthesis as well asmembrane structure and function are also magnesium-dependent. Levels ofmagnesium will affect neurotransmitter release including glutamate andacetylcholine release. It also regulates the activity of calciumtransporters and opening of the non-methyl-D-aspartate (NMDA) glutamatereceptors. Magnesium is known to have anti-oxidant, anti-apoptosis andto modulate lipid formation and transport. In addition to its cellulareffects, magnesium can modulate physiological functions involved inregulation of blood flow and edema development.

During the last decade, a number of studies have reported that brainlevels of free magnesium decline following TBI in animal model and inclinical settings. Decrease in brain levels of free magnesium from 40 to60% has been observed in various TBI animal models including the fluidpercussion model (Vink et al., 1991; Headrick et al., 1994), focalimpact model (Suzuki et al., 1997) as well as more diffuse models ofbrain injury (Heath and Vink, 1996; Smith et al., 1998). Furthermore, inthe rodent fluid percussion TBI model, a linear correlation wasestablished between changes in brain free magnesium levels, energeticpotential (phosphorylation potential) and functional (motor) outcomes(reviewed in Vink and Cernak, 2000). Decreases in magnesium levels havealso been reported in experimental spinal cord injury (Vink et al.,1989).

A direct correlation was also established in TBI patients between levelsof magnesium and the level of recovery (Mendez et al., 2005). TBIpatients as well as human suffering from acute ischemic and/orcerebrovascular events are more susceptible to develop a conditioncalled hypomagnesemia where availability of free magnesium is impaired(Polderman et al., 2000). Hypomagnesemia is also associated withincreased mortality in patients in general who require the attention ofthe intensive care unit (Chernow et al., 1989; Rubeiz et al., 1993).

Magnesium supplementation initiated from minutes to hours after onset ofCNS trauma showed neuroprotective effects in animal models of TBI (Heathand Vink, 1999; Esen et al., 2003; Vink et al., 2003; Feng et al., 2004and Turner et al., 2004), spinal cord injury (Suzer et al., 1999;Kaptanoglu et al., 2003) and stroke (Yang et al., 2000; Westermaier etal., 2003 and 2005).

Clinical evaluation of intravenous administration of magnesium sulfateup to 12 hours following stroke onset showed no significant improvementin a multicenter trial involving 2589 patients (Muir et al., 2004). Afollow-up study has been initiated to look at the potential effect of anearlier intervention where magnesium sulfate would be administeredwithin 2 hours of stroke onset (Saver et al., 2004). Another clinicaltrial initiated in 1999 at the University of Washington (Seattle) wasevaluating magnesium sulfate therapy for TBI patients and preliminarydata were also negative in this trial.

Magnesium supplementation has also been extensively studied in animalsand humans for its ability to reduce acute and chronic pain. However,mixed results have been reported from clinical trials evaluating theefficacy of magnesium (alone or in combination) in reducing painassociated with various surgical procedures (Bolcal et al., 2005; Apanet al., 2004; Bathia et al., 2004; McCartney et al., 2004), headache andacute migraine attacks (Cete et al., 2005; Corbo et al., 2001; Bigal etal, 2002), peripheral neuropathies (Brill et al., 2002; Felsby et al.,1996), cancer (Crosby et al., 2000), primary fibromyalgia syndrome(Moulin, 2001; Russel et al., 1995) and chronic limb pain (Tramer andGlynn, 2002). In addition, it appears that magnesium analgesic effectmay be of a short duration such as 4 hours or less (Crosby et al.,2000). Magnesium may also induce side effects such as flushing andaching that can reduce its therapeutic window (Tramer and Glynn, 2002).Magnesium supplementation therapies can be achieved by using varioussalts including magnesium sulfate, chloride, gluconate and magnesium-ATPleading to similar neuroprotective effects in animal models of CNSinjury (McIntosh et al., 1989; Izumi et al., 1991; Hoane et al., 2003;Turner et al., 2004; reviewed in Vink and McIntosh, 1990).

The inventors have found that administration of PEG alone or magnesiumalone had no effect on the loss of cognitive functions following braininjury whereas cognitive functions or more precisely, the ability tolearn a new spatial task, was improved by >30% in animals treated withboth PEG and magnesium solutions. Combined treatment with PEG andmagnesium was also significantly more potent than treatment with eithercomponent alone in animal models of SCI reducing the lesion size byhalf, improving locomotor recovery and reducing the occurrence ofneuropathic pain. In an acute model of tissue inflammation, the combinedPEG and magnesium solution was also more effective than PEG or magnesiumalone at reducing symptomatic pain. The discovery of a synergisticeffect between PEG, a biomembrane sealing agent, and magnesium is highlysignificant as it can lead to the development of therapeuticformulations with improved efficacy for the treatment of neuronaltrauma, inflammatory and painful conditions.

These results suggest that a biomembrane sealing agent, such as, forexample, PEG, may also potentiate the beneficial effects of othertherapeutic agents. In different embodiments, such bioactive agentsinclude, neurotransmitter and receptor modulators, anti-inflammatoryagents, antioxidants, anti-apoptotic agents, nootropic and growthagents; modulators of lipid formation and transport, electricalstimulation, blood flow modulators and any combinations thereof.

Suitable examples of antioxidants include, without limitation, freeradical scavengers and chelators enzymes, co-enzymes, spin-trap agents,ion and metal chelators, lipid peroxidation inhibitors such asflavinoids, N-tert-butyl-alpha-phenylnitrone, NXY-059, Edaravone,glutathione and derivates, and any combinations thereof.

Suitable examples of anti-inflammatory agents, include, withoutlimitation, steroids such as methyl prednisolone, Triamcinolone,modulators of an inflammatory cytokine such as IL-10, IL-1, IL-8,TNF-alpha and their receptors, COX inhibitor such as DFU and modulatorsof immune cell functions such as CD11b/CD18 antibody.

Suitable examples of neurotransmitter and receptor modulators include,without limitations, glutamate receptor modulators, cannabinoidreceptors modulators, and any combinations thereof. A person of ordinaryskill in the art will appreciate that one of the receptor modulators isa ligand naturally occurring in a subject's body. For example, glutamatereceptors modulators include glutamate.

In another embodiment, the at least one bioactive agent is a modulatorof glutamate transmission, such as (1S,2S)-1-(4-hydroxyphenyl)-2-(4-hydroxy-4-phenylpiperidino)-1-propanol(also known as CP-101,606), Riluzole (Rilutek®), Topiramate, Amantadine,Gacyclidine, BAY-38-7271, S-1749, YM872 and RPR117824.

In another embodiment, the at least one bioactive agent is a cannabinoidreceptor modulator such as dexanabinol (Pharmos Corporation, Iselin,N.J., USA).

Anti-apoptotic agents include, without limitations, inhibitors ofpro-apoptotic signals (e.g., caspases, proteases, kinases, deathreceptors such as CD-95,modulators of cytochrome C release, inhibitorsof mitochondrial pore opening and swelling); modulators of cell cycle;anti-apoptotoc compounds (e.g., bcl-2); immunophilins includingcyclosporine A, minocycline and Rho kinase modulators, and anycombinations thereof. Suitable non-limiting examples of Rho pathwaymodulators include Cethrin, which is a modified bacterial C3 exoenzyme(available from BioAxone Therapeutics, Inc., Saint-Lauren, Quebec,Canada) and hexahydro-1-(5-isoquinolinylsulfonyl)-1H-1,4-diasepine (alsoknown as Fasudil, available from Asahi Kasei Corp., Tokio, Japan).

Nootropic and growth agents include, without limitation, growth factors;inosine, creatine, choline, CDP-choline, IGF, GDNF, AIT-082,erythropoietin, Fujimycin (IUPAC name [3S-[3R*[E(1S*,3S*,4S*)],4S*,5R*,8S*,9E,12R*,14R*,15S*,16R*,18S*,19S*,26aR*]]-5,6,8,11,12,13,14,15,16,17,18,19,24,25,26,26a-hexadecahydro-5,19-dihydroxy-3-[2-(4-hydroxy-3-methoxycyclohexyl)-1-methylethenyl]-14,16-dimethoxy-4,10,12, 18-tetramethyl-8-(2-propenyl)-15,19-epoxy-3H-pyrido[2,1-c][1,4]oxaazacyclotricosine-1,7,20,21(4H,23H)-tetrone, monohydrate, also known as FK-506 and anycombinations thereof.

Suitable non-limiting examples of modulators of lipid formation,storage, and release pathways are apolipoprotein; statins; and anycombinations thereof.

Suitable non-limiting of blood flow modulators are adenosine receptormodulators such as ATL-146e and agents that modulate new vesselformation such as CM101.

In yet another embodiment, the at least one bioactive agent is anelectrical or magnetic stimulation. Electrical or magnetic stimulationmay be delivered from a site adjacent to the site of a pathologicalcondition (e.g., trauma). For example, if the pathological condition isa spinal injury at the level of C-6,the electrical or magneticstimulation may be delivered one segment above and one segment below thetrauma (i.e., C-5 and C-7).

A person of ordinary skill in the art will appreciate that multiplesources exist for delivering the electrical or magnetic stimulation. Inone embodiment, the source is an Oscillating Field Stimulator (OFS) asdescribed, for example in Shapiro, J. Neurosurg. Spine, 2: 3-10 (2005),incorporated herein by reference in its entirety. Briefly, the outsidecase of the OFS can be made of materials known to be safe for humanapplications, such as, for example, a fluoropolymer and a siliconesealant. Inside the case are the power block, timing/switching block,current regulation block, and fail-safe device. The power block providesthe direct-current power source for the unit involving a single 3.6-Vorganic lithium battery with a rated capacity of 2400 mAmp/hour. Thetiming/switching block includes a complementary metal oxidesemiconductor 14-stage binary ripple counter device with an onboardoscillator timed for 15-minute intervals along with a single-poledouble-throw analog switch. A fail-safe semiconductor chip is programmedto shut down the OFS if the power falls to 2.6 V, if there is a failureto oscillate, or if there are current changes indicative of an internalshort circuit. Current regulation can be set by another semiconductordevice that delivers 200 μAmp to each pair of electrodes for a totalcurrent of 600 μAmp. The electrodes can be made of standard pacemakercable and a platinum/iridium tip with a 4.72-mm² surface area. Amagnet-controlled reed switch can be used to turn the device on or off.When a magnet is on the switch, the device is turned off. When the unitis turned on, it delivers a field of 500 to 600 μV/mm and a currentdensity of 42.4 μAmp/mm² for each electrode.

Thus, in one embodiment of the invention the total current of electricalor magnetic stimulation may be between about 400 μAmp and about 700μAmp, or between about 450 μAmp and about 650 μAmp, or between about 500μAmp and about 600 μAmp. The current density of the electrical ormagnetic stimulation may be between about 30 μAmp/mm² and about 50μAmp/mm², between about 40 μAmp/mm² and about 45 μAmp/mm², or about 43μAmp/mm².

In another embodiment, a transcutaneous electrical nerve stimulation(TENS) may be used as the at least one bioactive agent. A person ofordinary skill in the art will appreciate that one advantage of TENS isthat it is non-invasive and that the guidelines for TENS are provided,for example, in Resende et al., Eur. J. Pharmacol. 504:217-222 (2004),incorporated herein by reference in its entirety. Conveniently, thepractitioner may use equipment which is commercially available, such as,for example, a Neurodyn III apparatus (IBRAMED, Brazil).

If TENS is selected as the at least one bioactive agent of choice, indifferent embodiments of the invention, the electrical stimuli may bereleased with a frequency of between about 4 Hz and about 130 Hz,wherein a duration of an individual electrical stimulus is between about60 and about 200 μs, or between about 100 and about 160 μs or betweenabout 125 and 135 μs.

Thus, a combined treatment comprising an administration of a biomembranesealing agent, such as for example, one of the polymers disclosed above,and at least one bioactive agent, such as, for example, a magnesiumcompound, has a positive and synergistic effect in reducing the lesionsize, improving functional recovery and reducing chronic pain followingneuronal trauma as well as reducing acute pain linked to tissueinflammation.

Accordingly, in one aspect, the invention comprises a pharmaceuticalcomposition comprising at least one biomembrane sealing agent and atleast one bioactive agent. As discussed above, in one embodiment, the atleast one active agent comprises at least one magnesium compound.

A person of ordinary skill in the art will undoubtedly recognize that atleast one magnesium compound may be just about any molecule providing asource of magnesium ions, such as, for example, a magnesium salt. In apreferred embodiment, the magnesium salt is non-toxic. Suitablenon-limiting examples of the at least one magnesium compound includemagnesium sulfate, magnesium chloride, magnesium gluconate, magnesiumATP, and any combination thereof.

A person of ordinary skill in the art will also appreciate that at leastone marker may be included into the pharmaceutical composition of thepresent invention. For example, the at least one marker may comprise anymolecule or a cocktail of ingredients distribution of which is easy tovisualize and monitor. Thus, in one embodiment, the at least one markermay be a radiographic marker, such as for example, barium, calciumphosphate, and metal beads. In another embodiment, the at least onemarker may comprise iodine-based contrast agents, such as, for example,iopamidol, commercially available as Isovue™ (Bracco Diagnostics Inc.,Princeton, N.J.) or iodixanol, commercially available as Visipaque™(Nyocomed, Inc., Princeton, N.J.), and gandolinium-based contrastagents, such as, for example, gadodiaminde, commercially available asOmniscan™ (available from GE Healthcare, Princeton, N.J.).

Therapeutic Formulations

Therapeutic formulations comprising the pharmaceutical composition ofthe present invention can be prepared for storage by mixing the at leastone biomembrane sealing agent and the at least one bioactive agent withoptional physiologically acceptable carriers, excipients or stabilizers(see, e.g., Remington's Pharmaceutical Sciences 16th edition, Osol, A.Ed. (1980)), in the form of lyophilized formulations or aqueoussolutions. Acceptable carriers, excipients, or stabilizers are nontoxicto recipients at the dosages and concentrations employed, and includebuffers such as phosphate, citrate, and other organic acids;antioxidants including ascorbic acid and methionine; preservatives (suchas octadecyldimethylbenzyl ammonium chloride; hexamethonium chloride;benzalkonium chloride, benzethonium chloride; phenyl, butyl or benzylalcohol; alkyl parabens such as methyl or propyl paraben; catechol;resorcinol; cyclohexanol; 3-pentanol; and m-cresol); low molecularweight (less than about 10 residues) polypeptide; proteins, such asserum albumin, gelatin, or immunoglobulins; amino acids such as glycine,glutamine, asparagine, histidine, arginine, or lysine; monosaccharides,disaccharides, and other carbohydrates including glucose, mannose, ordextrins; chelating agents such as EDTA; sugars such as sucrose,mannitol, trehalose or sorbitol; salt-forming counter-ions such assodium; and/or metal complexes (e.g., Zn-protein complexes).

The formulations herein may also contain more than one active compoundas necessary for the particular indication being treated, preferablythose with complementary activities that do not adversely affect eachother. Such molecules are suitably present in combination in amountsthat are effective for the purpose intended.

The at least one bioactive agent and/or the at least one biomembranesealing agent may also be entrapped in a microcapsule prepared, forexample, by coacervation techniques or by interfacial polymerization,for example, hydroxymethylcellulose or gelatin-microcapsule andpoly-(methylmethacylate) microcapsule, respectively, in colloidal drugdelivery systems (for example, liposomes, albumin microspheres,microemulsions, nano-particles and nanocapsules) or in macroemulsions.Such techniques are disclosed in Remington's Pharmaceutical Sciences16th edition, Osol, A. Ed. (1980).

Sustained-release preparations may also be prepared. Suitable examplesof sustained-release preparations include semipermeable matrices ofsolid polymers containing the at least one biomembrane sealing agentand/or the at least one bioactive agent, which matrices are in the formof shaped articles, e.g., films, or microcapsule. Examples ofsustained-release matrices include, without limitations, polyesters,hydrogels (for example, poly(2-hydroxyethyl-methacrylate), orpoly(vinylalcohol)), polylactides (see, e.g., U.S. Pat. No. 3,773,919),copolymers of L-glutamic acid and y ethyl-L-glutamate, non-degradableethylene-vinyl acetate, degradable lactic acid-glycolic acid copolymerssuch as the LUPRON DEPOT™ (injectable microspheres composed of lacticacid-glycolic acid copolymer and leuprolide acetate), andpoly-D-(−)-3-hydroxybutyric acid. Polymers such as ethylene-vinylacetate and lactic acid-glycolic acid enable release of molecules forover 100 days.

A person of ordinary skill in the art will undoubtedly appreciate thatthe at least one biomembrane sealing agent may be included into or usedas the semipermeable matrix. In this embodiment, both the at least onebiomembrane sealing agent and the at least one bioactive agent arereleased as the semipermeable matrix degrades.

Further, a person of ordinary skill in the art will recognize that theat least one biomembrane sealing agent and/or the at least one bioactiveagent may be implanted into the subject, for example, in forms of a pumpor a depot. A suitable non-limiting design of a depot implant isdiscussed in details in a co-pending application Ser. No. 11/403,373entitled Drug Depot Implant Designs And Methods Of Implantation, filedon Apr. 13, 2006.

A person of ordinary skill in the art will recognize that the use of theat least one marker is especially advantageous in combination with thisembodiment of the invention. The at least one marker may be included onthe drug depot implant itself. In this embodiment, a practitioner willbe better equipped to accurately position the implant into a tissue of apatient. As discussed above, the at least one marker may be aradiographic marker, such as, for example, barium, calcium phosphate,and metal beads. In another embodiment, the at least one marker maycomprise iodine-based contrast agents, such as, for example, iopamidol,commercially available as Isovue™ (Bracco Diagnostics Inc., Princeton,N.J.) or iodixanol, commercially available as Visipaque™ (Nyocomed,Inc., Princeton, N.J.), and gandolinium-based contrast agents, such as,for example, gadodiaminde, commercially available as Omniscan (availablefrom GE Healthcare, Princeton, N.J.). Such markers will also permit thepractitioner to track movement and degradation of the implant in thetissue over time. In this embodiment of the invention the practitionermay accurately position the implant in the tissue using any of thenumerous diagnostic imaging procedures known to one of ordinary skill inthe art. Such diagnostic imaging procedures include for example, X-rayimaging or fluoroscopy.

In another embodiment, the at least one biomembrane sealing agent and/orthe at least one bioagent may be administered locally via a catheterpositioned at or near a site of the pathological condition, e.g.,neuronal injury. In this embodiment, the catheter has a proximal end anda distal end, the proximal end having an opening to deliver the at leastone biomembrane sealing agent and/or the at least one bioagent in situ,the distal end being fluidly connected to a pharmaceutical deliverypump. For example, the proximal end of the catheter delivers the atleast one biomembrane sealing agent and/or the at least one bioagentwithin 10 cm of the site of the pathological condition, moreparticularly, within 5 cm of the site of the pathological condition, andeven more particularly, within 1 cm of the site of the pathologicalcondition. The catheter may be positioned via a minimally invasiveprocedure, such as, for example, by accessing a blood vessel adjacent orsupplying blood to the site of the pathological condition.

It would be within the expertise of a person of ordinary skill in theart to recognize that the at least one biomembrane sealing agent and theat least one bioactive agent may be delivered independently of eachother. In one non-limiting example, the at least one biomembrane sealingagent may be delivered through an intramuscular injection and the atleast one bioactive agent is delivered via an implant. A person ofordinary skill in the art will undoubtedly recognize that a large numberof combinations is possible.

A person of ordinary skill in the art will further recognize that insome cases it may be advantageous to ship and store the at least onebiomembrane sealing agent and the at least one bioactive agentseparately and pre-mix these compounds at a desired time, e.g., one hourprior to administration, or even to administer those compounds withoutpre-mixing. Accordingly, in another aspect, the invention provides a kitcomprising at least one biomembrane sealing agent, at least onebioactive agent, and a set of instructions comprising information onmaking an injectable composition, comprising more than about 10% of theat least one biomembrane sealing agent. In one embodiment of theinvention, the composition is incapable of forming a gel.

A person of ordinary skill in the art will further recognize that thekit provides a practitioner with an advantageous flexibility inselecting the ratios of the at least one biomembrane agent and the atleast one bioactive agent.

A person of ordinary skill in the art will appreciate that the set ofinstruction may be provided in any medium, including, withoutlimitations, printed, audio and video recorded, and electronic.

In another aspect, the invention provides a method of treating apathological condition, the method comprising delivering to a subject inneed thereof a therapeutically effective amount of at least onebiomembrane sealing agent and a therapeutically effective amount of atleast one bioactive agent, wherein the at least one biomembrane sealingagent is delivered in an injectable composition, wherein the at leastone biomembrane sealing agent comprises more than about 10% of theinjectable composition. In one embodiment of the invention, thecomposition is incapable of forming a gel. In different embodiments, thepathological condition is selected from the group consisting of neuronalinjury, tissue injury, surgical intervention, inflammation, and anycombination thereof.

Examples of suitable pathological conditions include, withoutlimitations, metabolic neuropathies such as diabetic and alcoholicneuropathies, postherpetic neuralgia, trauma to the central nervoussystem such as stroke, traumatic brain, spinal cord or cauda equineinjuries, pain derived from mechanical or biochemical neuronal insultssuch as carpal tunnel syndrome, phantom limb pain and symptomatic painassociated with degenerative conditions such as multiple sclerosis,arthritis and other joint diseases, persistent symptomatic pain derivedfrom surgical or other invasive interventions as well as chronic painderived from injury of peripheral neuronal or non-neuronal tissues.

The therapeutically effective amount of at least one biomembrane sealingagent and the therapeutically effective amount of at least one bioactiveagent may be delivered independently of each other by an intravenousadministration, an intramuscular administration, intrathecaladministration, subcutaneous administration, epidural administration,intra-articular administration, parenteral administration, directapplication onto or adjacent to a site of the pathological condition,and any combinations thereof. Initiation of individual treatments couldbe separated by a few hours, e.g., up to about 24 hours, or morepreferably, up to about 16 hours, or more preferably of up to about 8hours, or even more preferably, up to 4 hours. Thus, the at least onebiomembrane sealing agent and the at least one bioactive agent may bedelivered from independent sources and/or by different methods, or theymay be mixed prior to delivery.

A person of ordinary skill in the art will further recognize thatcertain invasive procedures, such as, for example, brain or spinal cordsurgeries, leave a subject with a neuronal injury. Accordingly, in oneembodiment of the invention, the at least one biomembrane sealing agentand/or the at least one bioactive agent is delivered to the subjectprior to the event triggering the occurrence of the pathologicalcondition. In one non-limiting example, the event is brain surgery andthe pathological condition is an injury to CNS neurons.

Specific embodiments according to the methods of the present inventionwill now be described in the following non-limiting examples.

EXAMPLES Example 1 Treatment with a Combination of PEG and MagnesiumCompound but not with PEG Alone or with Magnesium Compound AloneImproved Cognitive Functions Following TBI

Animals

Male Sprague-Dawley rats (Harlan Sprague-Dawley, Indianapolis, Ind.),weighing 350-450 grams each were given free access to food and waterbefore the experiment. The animals were anesthetized with halothane (1%in 70%/30% NO₂/O₂ by mask).

Brain temperatures were monitored using a rectal thermometer. Theanimals' body temperature was maintained at 37° C. by using awater-jacketed heating pad. Brain temperature was monitored for 1 hourprior to injury to 6 hours following injury and was recorded at30-minute intervals.

The controlled cortical impact model of brain injury utilized in thisstudy has been described in detail (Scheff and Sullivan, 1999). Animalswere anesthetized with isoflorane and placed in a stereotaxic frame inthe supine position. A 6 mm craniotomy located approximately midwaybetween bregma and lambda, just above the somatosensory cortex, was madeusing a Michele trephine. The skull disk was then removed withoutdisturbing the underlying dura mater. The exposed brain was injuredusing an electronically controlled piston (5 mm diameter, 65 kdynes).The animals in the “sham” group underwent identical surgical procedures,but the surface of the brain was not impacted.

Following surgery, animals were placed in their home cage and dosing wasinitiated. Animals were treated with saline (Group 1: sham, Group 2:injured), PEG (Group 3: injured), magnesium (Group 4: injured) or acombination of PEG and magnesium solutions (Group 5: injured). The PEGsolution was composed of PEG3350 at 30% in 0.5% saline (custom made byAAIPharma Developmental Services, Wilmington, N.C.). The magnesiumsolution was composed of magnesium sulfate at 50% in an injectableformulation (American Regent Laboratories, inc). The magnesium solutionwas diluted in saline 1:1 before intravenous injection as recommended bythe manufacturer. In the cases of combined PEG+Mg combined treatments,animal received the PEG and magnesium components from two individualsolutions.

In more details, each animal received 2 injections:

-   -   Group 1: Sham—1.23 ml/kg of saline solution followed by 1.23        ml/kg of saline solution;    -   Group 2: Injured—1.23 ml/kg of saline solution followed by 1.23        ml/kg of saline solution;    -   Group 3: Injured—2.33 ml/kg of PEG solution followed by 0.12        ml/kg of saline solution;    -   Group 4: Injured—0.12 ml/kg of magnesium solution followed by        2.33 ml/kg of saline solution;    -   Group 5: Injured—2.33 ml/kg of PEG solution followed by 0.12        ml/kg of magnesium solution.

The first two injections were administered 15 minutes following injury.Another round of two injections was given 6 hours later. The singledoses of PEG and magnesium sulfate administered were of 0.7 g/kg and0.115 mmol/kg and total dose of 1.4 g/kg and 0.230 mmol/kg (body weight)respectively. The compounds tested were administered by bolusintravenous injection. There were 10 animals per group. These studieswere performed in a randomized and blinded fashion such as the solutionswere sent to the Research Center in blindly labeled packages and thecode was not revealed to the Research Center before the end of thestudy.

Assessment of Cognitive Function

The effects of PEG, magnesium sulfate and combined treatment on recoveryof cognitive function following TBI were evaluated using the MorrisWater Maze Test. This behavioral endpoint measure the ability of the ratto learn a new task based on the memorization of spatial cues. It is themost widely utilized behavioral test to assess functional recoveryfollowing TBI in rats. Briefly, the open-field circular pool was of 127cm in diameter×56 cm in height, with a removable circular plasticplatform 13.5 cm in diameter (Morris, 1984). The pool and platform werepainted flat black in color, and the water was colored black withnon-toxic black powdered paint to obscure the platform location.Platform location during training trials was always in the South-Eastquadrant, approximately 30 cm from the pool wall, and 1 cm below thesurface of the water (19-21° C.). Spatial cues located in the testingroom remained constant throughout testing. Each day of testing consistedof four 60-s trials to navigate to the hidden platform. Once theplatform was located, animals were allowed a 15-s platform sit followedby a 5-min inter-trial interval (ITI). If at the end of 60 s the rat wasunable to find the platform, the subject was guided to the platform andallowed a 15-s platform sit, followed by a 5-min ITI. Entry into thewater maze was randomized between one of four locations (North, South,East, West) for the training tests each day. Five minutes following thefourth acquisition trial on the fifth day, the platform was removed fromthe pool and a 30-s probe trial was performed; pool entry during theprobe trial was always from the NW location. A video camera (Sony, CCTVCamera) was located directly above the pool to record swimming duringtraining and probe tests. The animals were tested from Day 9-14following injury and treatment.

To evaluate the significance of difference between experimental groups,data were analyzed using an unpaired, two-tailed t statistical test withconfidence intervals of 95%.

The rats in the TBI groups were subjected to anesthesia, craniotomy andcontrolled cortical impact delivered directly on the dura. The “sham”group underwent identical surgical procedures, but the surface of thebrain was not impacted. Following brain injury, the rats were treatedwith intravenous injections of saline, PEG, Magnesium or combined PEGand Magnesium solutions. All rats received two injection regiments, onefifteen minutes post-injury and another one 6 hours later. From day 9 today 14 post-injury, the animals were tested for their ability to learn anew task by using the Morris Water Maze. Briefly, the animal isevaluated for its ability to find a hidden platform in a circular poolby using visual cues located on the 4 walls surrounding the pool. Eachtesting day (day 9-14), the animals are allowed 4 training trialsfollowed by a probe trial. The time required to locate the platform orlatency (sec) during the probe trial is reported here.

Following craniotomy only (sham group) or craniotomy and controlledcortical impact (TBI groups), rats were treated with intravenousinjections of saline, PEG or Magnesium (Mg) solutions. Treatment withPEG alone or Magnesium alone had no effect on cognitive functionsfollowing TBI. Combined PEG and Magnesium (Mg) treatments significantlyimproved recovery of cognitive functions between days 12-14 post-TBI,reducing the time to locate the platform by approximately 30% relativeto animals treated with saline only (p<0.05). At day 14 post-injury,combined PEG+Mg treatments also showed a positive and synergistic effectrelative to PEG alone treatment (p<0.0001) or Mg alone treatment(p=0.0001).

Example 2 Treatment with a Combination of PEG and Various MagnesiumCompounds are Equally Effective in a Model of TBI

Following the same experimental procedure presented in example 1, TBIanimals were treated with saline, PEG, magnesium chloride orPEG+magnesium chloride. TBI animals received 2 injections, one injection15 minutes post-TBI and the second injection 6 hours later. The totaldoses were 1.4 g/kg PEG and 0.230 mmol/kg magnesium chloride.

Treatment with PEG or magnesium chloride had no significant effect oncognitive recovery after TBI. Combined PEG+magnesium chloride treatmentssignificantly improved recovery of cognitive functions between days10-14 post-TBI, reducing the time to locate the platform byapproximately 30% (as seen for PEG+magnesium sulfate in example 1)relative to animals treated with saline only (p<0.01).

Example 3 Treatment with a Combination of Magnesium and PEG, where PEGis used at Concentrations form 15-30% are Effective in a Model of TBI

Following the same experimental procedure presented in example 1, TBIanimals were treated with saline, PEG 15%+Magnesium chloride, PEG20%+Magnesium chloride, PEG 30%+Magnesium chloride. TBI animals received2 injections of 3.33 ml/kg, one injection 15 minutes post-TBI and thesecond injection 6 hours later. For all 3 solutions, the total dose ofmagnesium chloride was 0.230 mmol/kg.

In this TBI study, cortical lesion volume was also assessed. Tissuesections were stained with Cresyl Violet and the volume of the corticallesion will be determined. Twelve equally spaced sections (1 mm) wereevaluated using a video-based image analysis system (NIH Image). Thelesion volume in each section were determined with a computer-assistedimage analysis system, consisting of a Power Macintosh computer equippedwith a Quick Capture frame grabber card, Hitachi CCD camera mounted onan Olympus microscope and camera stand. NIH Image Analysis Software, v.1.55 was used. The images were captured and the total area of damagedetermined over the twelve sections. A single operator blinded totreatment status performed all measurements. The total cortical area wasmeasured on the ipsilateral side of the brain using lamina 1 and thecorpus callosum as boundaries. The area of the cavity produced by thelesion was also calculated for each tissue section. Lesion volume wascalculated by multiplying the total length of the cortex analyzed by theaverage volume of the cortex.

Treatment with combined solutions containing magnesium chloride and PEGat 15, 20 and 30% improved recovery of cognitive functions between days10-14 post-TBI, reducing the time to locate the platform byapproximately 19, 32 and 56%, respectively after TBI.

In addition, treatment with combined solutions containing magnesiumchloride and PEG at 15, 20,and 30% reduced the lesion volume by 6,29,and 47%.

Example 4 Treatment with a Combination of PEG and Magnesium Compound butnot with PEG Alone or with Magnesium Compound Alone Improved MotorFunctions Following SCI in a Dural Impact Model

Animals

Sprague-Dawley female rats (200-225 gm) received a spinal cord contusionusing the Precision Scientific Inc pneumatic impactor. Prior to surgery,rats were assigned to different treatment groups based on a randomizedblock design so that on any given surgery day all treatments would beincluded. The rats were anesthetized with ketamine (80 mg/kg) andxylazine (10 mg/kg) before a laminectomy will be performed at the10^(th) thoracic vertebra (T₁₀). The vertebral column was stabilizedwith angled clamps on the upper thoracic (T8) and lumbar (T11) levelsand the impactor with a tip diameter of 2 mm was delivered atapproximately 50 kd onto the exposed, intact dura overlying the dorsalspinal cord. The impactor was immediately removed, the wound irrigatedwith saline, and the muscle and skin openings sutured together. Femaleanimals were used due to the paralysis associated with the injury andease of voiding the bladder.

Following surgery, animals were placed in their home cage and dosing wasinitiated. Animals were treated with saline (Group 1: sham, Group 2:injured), PEG (Group 3: injured), magnesium (Group 4: injured) or acombination of PEG and magnesium solutions (Group 5: injured). The PEGsolution was composed of PEG3350 at 30% in 0.5% saline (custom made byAAIPharma Developmental Services, Wilmington, N.C.). The magnesiumsolution was composed of magnesium sulfate at 50% in an injectableformulation (American Regent Laboratories, inc). The magnesium solutionwas diluted in saline 1:1 before intravenous injection as recommended bythe manufacturer. In the cases of combined PEG+Mg combined treatments,animal received the PEG and magnesium components from two individualsolutions.

In more details, each animal received 2 injections:

-   -   Group 1: Sham—1.79 ml/kg of Saline followed by 1.79 ml/kg of        Saline;    -   Group 2: Injured—1.79 ml/kg of Saline followed by 1.79 ml/kg of        Saline;    -   Group 3: Injured—3.33 ml/kg of PEG solution followed by 0.24        ml/kg of Saline;    -   Group 4: Injured—0.24 ml/kg of magnesium solution followed by        3.33 ml/kg of Saline;    -   Group 5: Injured—3.33 ml/kg of PEG solution followed by 0.24        ml/kg of magnesium solution.

The first two injections were administered 15 minutes following injury.Another round of two injections was given 6 hours later. The total doseof PEG and magnesium sulfate administered was of 2 and 0.12 g/kg (bodyweight) respectively. The compounds tested were administered by bolusintravenous injection. There was 9-10 animals/group. These studies wereperformed in a randomized and blinded fashion such as the solutions weresent to the Research Center in blindly labeled packages and the code wasnot revealed to the Research Center before the end of the study.

Assessment of Motor Function

To assess locomotor recovery after SCI, animals were tested prior tosurgery and up to 6 weeks post-injury. Animals were placed in an openfield chamber (120 cm diameter, 25 cm wall height) for 4 minutes toassure that all subjects obtained a maximum score of 12 using a modifiedversion of the Basso, Beattie, and Bresnahan (BBB) locomotor ratingscale (Ferguson et al., 2004). Rats were placed in the open field for 4minutes and videotaped for scoring.

The rats in the SCI groups were subjected to anesthesia, laminectomy andcontrolled spinal impact delivered directly on the dura. The “sham”group underwent identical surgical procedures, but the surface of thespinal cord was not impacted. Following spinal cord injury, rats weretreated with intravenous injections of saline, PEG, Magnesium orcombined PEG and Magnesium solutions. All rats received two injectionregiments, one fifteen minutes post-injury and another one 6 hourslater. For the following 6 weeks post-injury, the animals were testedfor their locomotor ability using a modified version of the Basso,Beattie, and Bresnahan (BBB) locomotor rating scale with a maximum scoreof 12 (Ferguson et al., 2004).

To evaluate the significance of difference between experimental groups,data were analyzed using an unpaired, two-tailed t statistical test withconfidence intervals of 95%. Following laminectomy only (sham group) orlaminectomy and controlled impact (SCI groups), the rats were treatedwith intravenous injections of saline, PEG or magnesium (Mg) solutions.Treatment with PEG alone or Mg alone had no effect on motor functionsfollowing SCI. In contrast, combined PEG and Mg treatments significantlyimproved locomotor recovery as evaluated by the 12-point BBB scorerelative to SCI-Saline group.

At the end of the study, the spinal cords were extracted and fixed in 4%paraformaldehyde. For analysis, 20 um cryosections were stained foreriochrome cyanine (EC) to differentiate between white matter and cellbodies to calculate the amount of spared tissue through the lesion site.Tissue sparing was determined by computed image analysis be from 10evenly spaced sections through the injured T10 segment. The area ofnecrotic tissue was divided by the total cross-sectional area, convertedto a percentage and subtracted from 100%.

Animals were sacrificed on day 14 and spinal cord tissues were processedto determine the lesion volume. Treatment with PEG reduced the lesionvolume by 20% but this change was not statistically significant relativeto the control group (SCI-Saline). Animals treated with Mg showed asignificant reduction (33%) of the lesion volume as compared to controlanimals. Combined treatment with PEG and Mg solutions showed a positiveand additive effect significantly decreasing the lesion volume by 51%relative to control group. Average lesion volume in the animal groupthat received the combined treatments was significantly reduced relativeto animals treated with PEG alone (p=0.0393) or Mg alone (p=0.0364).

Example 5 Improved Motor Recovery Following Treatment with a Combinationof PEG and Magnesium Compound Relative to PEG alone or MagnesiumCompound Alone in a SCI Dural Compression Model

Animals

Wistar female rats weighing 200-250 grams received a controlledclip-compression injury at T4.This experimental model has beenpreviously described in details in Gris et al., 2004. Briefly, animalswere premedicated with diazepam (3.5 mg/kg, i.p.) and atropine (0.05mg/kg, s.c.) in order to facilitate a smooth induction of anesthesia by4% halothane and maintenance with 1.0-1.5% halothane. The T4 spinal cordsegment was exposed by dorsal laminectomy and a modified aneurysm clip,calibrated to 50 grams compression was passed extradurally around thecord and spring released for 60 seconds. Nerve roots were not disruptedduring the clip compression.

Following SCI, animals were placed in their home cage and dosing wasinitiated. Animals were treated with saline, PEG, magnesium or combinedPEG+magnesium solutions. The PEG solution was composed of PEG3350 at 30%in 0.5% saline (custom made by AAIPharma Developmental Services,Wilmington, N.C.). The magnesium solution was composed of magnesiumsulfate at 50% in an injectable formulation (American RegentLaboratories, inc) further diluted in saline 0.5%. For the combinedtreatment, PEG and magnesium solutions were pre-mixed a few minutesbefore administration. Treatment solutions were administered byintravenous injections.

Each animal received 2 injections, the first one 15 minutes after injuryand the second injection 6 hours later. The total dose of PEG andmagnesium sulfate administered was of 2 and 0.6 g/kg (body weight)respectively. There was 6-10 animals/group. These studies were performedin a randomized and blinded fashion such as the solutions were sent tothe Research Center in blindly labeled packages and the code was notrevealed to the examiners before the end of the study.

Assessment of Locomotor Recovery

Locomotor recovery was assessed by the 21-point Basso, Beatie andBresnahan (BBB) open field locomotor test (Basso et al., 1995), from day3-21 after injury. Locomotor function was evaluated by 2 blindedinvestigators at each testing period.

To evaluate the significance of difference between experimental groups,data were analyzed using an unpaired, two-tailed t statistical test withconfidence intervals of 95%. The Welch's modification was also used toanalyze the motor recovery to take in account the significantvariability in the control group.

Following SCI, rats were treated with saline, PEG, Mg or a combinedsolution of PEG and Mg. Locomotor recovery was assessed by the 21-pointBBB score system from day 3-25 post-injury. In this particular paradigm,PEG had no significant effect on motor recovery within the first 3 weeksafter injury. Some improvement was seen at day 7 post-SCI derived fromMg treatment. Animals that received the combined PEG+Mg treatment showedfaster recovery with an average BBB score significantly higher than PEGor Mg groups at day 14 post-injury.

Example 6 Treatment with a Combination of PEG and Magnesium Compound butnot with PEG Alone or with Magnesium Compound Along ReducedSeverity/Occurrence of Neuropathic Pain

Animals

Animals were prepared as described in Example 3.

Assessment of Development of Neuropathic Pain

Animals were tested for the development of neuropathic pain before andup to 3 weeks post-injury. Neuropathic pain occurrence and severity wasevaluated by the response of the animal to a stimulus that is normallynot painful or allodynia. Briefly, a modified Semmes-Weinsteinmonofilament (Stoelting Co, Wood Dale, Ill.) calibrated to generate aforce of 15 mN was applied to the dorsal trunk at dermatomescorresponding to spinal segments immediately rostral to the lesion level(T1-T3). The monofilament was applied 10 times for 3 seconds, with eachstimulus being separated by a 5 second interval, and the number ofavoidance responses out of a possible 10 were recorded. Avoidanceresponses were defined as flinching, escape, vocalization, or abnormalaggressive behaviors. The scoring system used to monitor mechanicalallodynia has been presented in details in Gris et al., 2004. Mechanicalallodynia testing was conducted twice per week and the 2 tests wereaveraged for each animal and reported as a weekly pain score.

To evaluate the significance of difference between experimental groups,data were analyzed using an unpaired, two-tailed t statistical test withconfidence intervals of 95%. The Welch's modification was also used toanalyze the motor recovery to take in account the significantvariability in the control group.

In general, the score of a non-injured animal would be equal to zero.The number of avoidance responses of the SCI animals injected withsaline increased progressively after injury to reach a mean pain scoreof 3.2±0.5 at week-3 post-injury consistent with the development ofneuropathic pain. In this particular paradigm, PEG or Mg had nosignificant effect on the development of neuropathic pain after SCI.Combined treatment with PEG+Mg dramatically reduced theoccurrence/severity of neuropathic pain to 1+/−0.6 and significantlylower than animals treated with saline.

Example 7 Treatment with a Combination of PEG and Magnesium Compound butnot with PEG Alone or with Magnesium Compound Alone Reduced Severity ofPain in a Model of Acute Inflammation

Animals

Animals were treated with saline (0.5% final), indomethacin, PEG,magnesium or combined PEG+magnesium solutions. Each animal received 2injections, the first one 1 hour following carrageenan injection and thesecond injection 6 hours later. The total dose of PEG and magnesiumchloride administered was of 2 and 0.135 g/kg (body weight)respectively. There were 10 animals per group. These studies wereperformed in a randomized and blinded fashion such as the solutions weresent to the Research Center in blindly labeled packages and the code wasnever revealed to the examiners.

Indomethacin (Fluka, cat. 57413, lot 450544/1) was dissolved in 25%hydroxyl-propyl beta cyclodextrin (hpBCD) by gentle heating andstirring. The PEG solution was composed of PEG3350 at 30% in 0.5%saline, the magnesium solution was composed of magnesium chloride at 2%and the combined PEG+magnesium solution was composed of 30% PEG3350+2%Magnesium Chloride (custom made by AAIPharma Developmental Services,Wilmington, N.C.).

Assessment of Pain

The carrageenan model (Iadarola et al., 1988) was used to induce hindpaw acute inflammation. Male Sprague-Dawley (258±2.2 g) wereanesthetized with isoflurane, and 50 μL of 2% λ-carrageenan (w/v; Sigma,catalog #C-3889,lot #122K1444) was injected intradermally into the lefthind paw using a 1 cc syringe fitted with a 27 g needle. For the shamgroup, 50 μL of 0.9% saline was injected into the hind paw in anidentical manner.

Twenty-four hours following carrageenan injection into the left paw, thesame paw was tested for mechanical allodynia. Baseline andpost-treatment values for non-noxious mechanical sensitivity wereevaluated using 8 Semmes-Weinstein filaments (Stoelting, Wood Dale,Ill., USA) with varying stiffness (0.4, 0.7, 1.2, 2.0, 3.6, 5.5, 8.5,and 15 g) according to the up-down method (Chaplan et al., 1994).Animals were placed on a perforated metallic platform and allowed toacclimate to their surroundings for a minimum of 30 minutes beforetesting.

To evaluate the significance of difference between experimental groups,data were analyzed using an unpaired, two-tailed t statistical test withconfidence intervals of 95%.

The mean pain score in the carrageenan-saline group was 3.4+/−0.5 andsignificantly lower (p<0.001) than the mean pain score of the sham(saline-saline) animals evaluated at 12.7+/−1.1.In thiscarrageenan-paradigm, treatment with PEG or Magnesium alone had nosignificant effect on the pain score relative to animals treated withsaline. Combined treatment with PEG+Mg significantly improved the painscore to 7.3+/−1.5 (p<0.05) relative to the carrageenan-saline group(3.4+/−0.5). PEG+Mg combined treatment was also significantly betterthan treatment with Mg alone (p<0.05). Indomethacin is a nonsteroidalanti-inflammatory drug that is approved by the FDA to relieve painassociated with inflammatory conditions. The combined PEG+Magnesiumformulation led to a better pain score (7.3+/−1.5) than the animalstreated with Indomethacin (5.6+/−0.9).

All patent and non-patent publications cited in this disclosure areincorporated herein in to the extent as if each of those patent andnon-patent publications was incorporated herein by reference in itsentirety. Further, even though the invention herein has been describedwith reference to particular examples and embodiments, it is to beunderstood that these examples and embodiments are merely illustrativeof the principles and applications of the present invention. It istherefore to be understood that numerous modifications may be made tothe illustrative embodiments and that other arrangements may be devisedwithout departing from the spirit and scope of the present invention asdefined by the following claims.

The invention claimed is:
 1. A method of reducing the volume of lesiondue to a spinal cord injury or a traumatic brain injury in a subject inneed thereof, the method comprising: a) delivering to the subject afirst injectable composition comprising magnesium chloride and 20-30%weight/volume of polyethylene glycol (PEG) in saline, wherein saidmagnesium chloride and PEG act synergistically to treat the injury; b)providing the dose of Mg in said magnesium chloride to be within a rangefrom 0.115 to 0.230 mmol of Mg/kg of the subject's weight and providingthe dose for PEG to be within a range from 0.7 to 1.4 g of PEG/kg of thesubject's weight for the first injectable composition; c) delivering tothe subject a second injectable composition comprising magnesiumchloride and 20-30% weight/volume of PEG in saline within 24 hours ofdelivering the first injectable composition wherein said magnesiumchloride and PEG act synergistically to treat the injury; d) providingthe dose of Mg in said magnesium chloride to be within a range from0.115 to 0.230 mmol of Mg/kg of the subject's weight and the dose forPEG to be within a range from 0.7 to 1.4 g of PEG/kg of the subject'sweight for the second injectable composition; and e) observing at least29% improvement in lesion volume over a similar dosage of Mgadministered alone in saline due to the synergistic effect ofco-administering Mg and PEG.
 2. The method of claim 1 wherein the PEGand the magnesium chloride are delivered by a method selected from thegroup consisting of an intravenous administration, an intramuscularadministration, an intrathecal administration, a subcutaneousadministration, an epidural administration, a parenteral administration,an intra-articular administration, a direct application or depositiononto or adjacent to a site of the injury, and any combinations thereof.3. The method of claim 2 wherein the method is intravenousadministration.
 4. The method of claim 1, wherein the PEG and magnesiumchloride are delivered independently of each other.
 5. The method ofclaim 1, wherein the PEG and the magnesium chloride are combined beforethe step of delivering to the subject in need thereof.
 6. The method ofclaim 1, wherein the PEG or the magnesium chloride are delivered from animplant.
 7. The method of claim 1 wherein the PEG or the magnesiumchloride are delivered from a pump.
 8. The method of claim 1, furthercomprising the step of delivering at least one marker with the PEG orthe magnesium chloride.
 9. The method of claim 1, wherein at least onecompound is delivered prior to an occurrence of the injury, said onecompound selected from the group consisting of the PEG and the magnesiumchloride.
 10. The method of claim 1, wherein the injectable compositionis incapable of forming a gel.